Biotechnology is, generally speaking, any technological application that uses biological systems (such as a complex microbiological community), living organisms (microorganisms, animals or plants) or their derivates (metabolites, proteins, nucleic acids) in the creation or modification of products or processes for specific uses. In this invention, we will focus on the biotechnology that employs microorganisms, especially biomining and microbiological bioremediation.
Many processes of microbiological biotechnology are carried out strengthening the development of the microbiological community originally present in the substrate to be treated, for instance the community present in ore in the case of biomining, or present in contaminated soil or water in the case of bioremediation. In these cases having a tool that that would allow us to evaluate the metabolic characteristics of the community present, would be of great use, for example, in judging whether the process we are interested in can be carried out with the community present, or if we should inoculate other adequate strains.
Up to now, the most common approach used in evaluating microbiological communities has been the identification of species present, by means of a variety of techniques such as selective cultures, fluorescent in situ hybridization (FISH), conjugated with specific markers such as detection probes, polymerase chain reaction (PCR) and macro and micro DNA arrays. Among the previously mentioned techniques, all the ones pertaining to molecular biology, that is to say FISH, detection probes, PCR, and macro and micro DNA arrays, are based on hybridizing the DNA of the unknown sample with known sequences that are specific for the microorganism or microorganisms that need to be detected or identified, for example, a certain specie or genus.
The problem with the previous approach is that there are many types of different microorganisms that may be present in these microbiological communities, so that many tests should be carried out to establish their composition, and then correlate these acquired data with the characteristics of each identified taxon, to finally establish whether the function of interest is present in the sample. If there were a microorganism not previously described, this correlation could not be carried out, even if it's metabolic activities were known, and even important for the process. So it rises the need for a method oriented for directly evaluating and controlling the metabolic characteristics of a microbiological community, and not for identifying what microorganisms it is composed of.
This technical problem has been resolved by means of the present invention, by creating an array of nucleotidic sequences for detecting and identifying genes that codify proteins with relevant activities in biotechnology present in a microbiological sample, and a Method for employing this array.
When changing the focus of the approach from the taxonomy of the studied community to the identification of proteins with relevant activities present in this community, we focus directly on the essential of the biotechnological process: the metabolic functions which ultimately carry out these processes. For example, each time the presence of Leptospirillum spp. is sought for in a bioleaching process, what really is being sought for is iron oxidization, and it is so for each of the microorganisms that are relevant in some biotechnological process.
The new approach, designed by us, makes it possible to respond to exactly what the operator of a biotechnological process requires: Does this community oxidize iron?, does it oxidize sulfur?, does it fix CO2?, does it fix nitrogen? With the approach that currently exists in the technique, the operator first knows what species are present, and then he or she must correlate this information with the characteristics of each of these species in order to answer the same questions.
In each case, we have designed fragments of DNA for identifying the genes that codify proteins with relevant activities in biotechnology that comply with an essential condition: the specificity of the mentioned fragment must correspond solely to the target gene. On the other hand, whenever it has been possible, regions have been sought which being specific for each target gene, and orthologically conserved. This is with the idea of detecting not only the genes that have been sequenced, but also genes that have not been described, that are orthologues of each target gene. The result of this search corresponds to fragments that are specific for a certain gene or function within a specific taxon in a specific region.
With these DNA fragments designed we have developed DNA fragment arrays which allow us to carry out the identification of the presence of these genes that codify proteins with relevant activities in biotechnology, which are present in a microbiological sample.
A good definition of DNA arrays is the one proposed by Schena and colleagues: “a microscopic and methodical arrangement of nucleic acids that allow simultaneous analysis of complex DNA samples” (Schena M., Heller, R. A., Theriault, P., Konrad, K., Lachenmeier, E. and Davis, R. W. Trends Biotechnol. 16, 301-306, 1998). Depending on the diameter of the printed DNA dot there are 2 types of arrays, macro arrays (300 microns or more), and micro arrays (less than 100 microns). The former can be made manually in the laboratory and the dots can be visualized without the help of special equipment. The latter require an automated printing system (normally a robotic printing platform) and specialized acquisition and image-processing equipment.
In the present invention, DNA fragment arrays include an ordered series of dots printed on a flat surface such as a sheet of glass, silicone or nylon, where each dot contains a large quantity of copies of a DNA fragment that is known and specific for a certain gene of interest in biotechnology.
The detection method that employs DNA fragment arrays includes simultaneous hybridization of the set of ‘dots’ of the array with an extract of labeled DNA from the sample under study. Normally, the DNA of the sample which has been labeled and in given case fragmented, is submitted to a denaturing stage in which the double strand of DNA separates, for example, with heat. As the temperature is lowered, the DNA, due to its physical-chemical characteristics, will tend to hybridize with its most exact complementary. When this DNA is in contact with the array, if there is a coincidence between the DNA of the sample and the fragment of DNA contained in the dot, the copies of the sample's labeled DNA will most probably remain linked specifically to that dot. This occurs due to the higher number of copies of complementary DNA contained in the dot of the array. In the image acquisition and processing stage of the hybridized array, this label will allow the detection of microorganisms present in the sample under study.
DNA labeling can be done with any known labeling technique, the most common of which are fluorescent or radioactive label.
The arrays and their use are known, and in the state of the art we find examples of arrays that detect the presence of microorganisms in a sample, but none of them focus on the detection of genes that codify proteins with relevant activities in biotechnology.
There are currently various protocols published for manufacturing DNA fragment arrays, as well as laboratories that render services in the manufacture of this kind of arrays. As a result, only the selection of the genes and the design of the fragments of DNA used, is what defines the specificity and usefulness of an array, because manufacture may vary in regard to the support, the method with which the fragments of DNA are bound to the support, the spatial distribution of the dots on this support, etc., depending on the company entrusted with the array manufacturing or the protocol employed for doing it in the laboratory itself. (Ye et al Journal of Microbiological Methods 47 (2001) 257-272).